Apolipoprotein B occurs naturally in two forms, apo-B100 and apo-B48, both encoded by the same gene. Apo-B100, a 550 KD protein, is the major protein responsible for cholesterol transport in the blood and plays a crucial role in cholesterol and lipoprotein metabolism. Apo-B100 is an integral component of very low density lipoproteins (VLDL) and intermediate density lipoproteins (IDL), and is the sole component of low density lipoprotein (LDL). Apo-B48, a 264 KD protein, is synthesized in the intestines of humans and rabbits and in the liver and intestines of rats and mice. In mice and rats, hepatic-derived apo-B48 is a component of VLDL; the total VLDL is a mixture of VLDL containing apo-B100 or apo-B48. Intestinally derived apo-B48 is secreted as an integral component of chylomicrons.
Apo-B48 is produced by a biological process in which the apoB primary transcript is postranslationally modified by a type of RNA processing known as RNA editing. The term RNA editing is used to describe the specific modification of mRNA (or the coding region of pre-RNA) that alters the genetic information encoded in the transcript.
Apolipoprotein B mRNA editing deaminates a specific cytidine (C.sup.6666) to create a uridine. This changes the codon at position 2153 from a genomically encoded CAA (glutamine) to an in-frame stop codon (UAA). Apolipoprotein B mRNA editing occurs in the small intestine of all mammals and in the liver of rats, mice, dogs, and horses.
Hepatic apo-B mRNA editing in the rat and mouse, both of which normally modify approximately 65% of the apoB mRNA, is developmentally and hormonally regulated. Editing activity is regulated by growth hormone, thyroxine, cortisol, fasting, and diet. Apolipoprotein B mRNA editing also demonstrates developmental regulation in the human intestine. Human fetal intestine at 11 weeks of gestation predominantly produces apo-B100, whereas at 16 weeks of gestation, both apo-B100 and apo-B48 are secreted in roughly equal proportions. In the adult intestine, only apo-B48 is secreted.
A specific 11 nucleotide "mooring" sequence in apoB mRNA occurring 5 nucleotides downstream from C.sup.6666 is critical for editing in vitro. When the mooring sequence is inserted into another location on apoB or non-apoB cDNA, the resulting chimeric RNA is edited in vitro (Driscoll et al. Mol. Cell. Biol. 13: 7288-7294 (1993); Backus et al. Biochim. Biophys. Acta. 1217: 65-73 (1994); Shah et al. J. Biol. Chem. 266: 16301-16304 (1991); Backus et al. Biochim. Biophys. Acta. 1219: 1-14 (1994)).
Several proteins appear to be necessary for apoB mRNA editing in vitro. One of these proteins has been cloned from a rat intestinal library (Teng et al. Science 260: 1816-1819 (1993)). This 27 kD protein, which deaminates cytidine.sup.6666 in apoB mRNA, has been designated APOBEC-1 (apoB mRNA-editing enzyme catalytic polypeptide #1) (Davidson et al. RNA 1:3 (1995)). The major functional domains of this RNA editing polypeptide are highly conserved in the cloned homologues of rat APOBEC-1 from human (Hadjiagapiou et al. Nucleic Acids Res. 22: 1874-1879 (1994); Lau et al. Proc. Natl. Acad. Sci. U.S.A. 91: 8522-8526 (1994)), rabbit (Yamanaka et al. J. Biol. Chem. 269: 21725-21734 (1994)), and mouse (Nakamuta et al. J. Biol. Chem. 270: 13042-13056 (1995)).
Transgenic mice and rabbits expressing APOBEC-1 have been generated (Yamanaka etal. Proc. Natl. Acad. Sci. U.S.A. 92: 8483-8487 (1995)). The transgenic mice and rabbits had liver dysplasia, and many developed hepatocellular carcinomas.
Comparison of gene expression in different tissues or under different conditions can be performed using the technique of differential mRNA display (Liang and Pardee, Science 257: 967-971 (1992)), also termed differential display reverse transcriptase PCR. In this technique, two or more RNA populations (e.g., RNA preparations from different tissues) are made into cDNA using reverse transcriptase and a set of oligonucleotide primers, one being anchored to the polyadenylate tail of a subset of mRNAs by, for example, the two nucleotide sequence CA, the other being short and arbitrary in sequence so that it anneals at different positions relative to the first primer. The resulting cDNA is amplified by PCR, using a 5' primer of arbitrary base sequence, chosen to anneal at positions randomly distributed in distance from the poly(A) tail. The resulting amplified DNA sequences can be separated by gel electrophosis. An amplified DNA band can be subcloned into a vector, or can be sequenced, for example, by using extended primers for futher amplification (Wang and Feuerstein, Biotechniques 18: 448-452 (1995)), ligation linked PCR (Reeves et al. Biotechniques 18: 18-20 (1995)).
Recent studies have provided some insight into the genetic basis for obesity. A mouse obesity gene, ob, and its human homologue have been cloned and sequenced (Zhang et al. Nature 372: 425-432 (1994)). Mutation in ob results in profound obesity and type II diabetes as part of a syndrome that resembles morbid obesity in humans (Friedman et al. Genomics 11: 1054-1062 (1991). Halaas et al. (Science 269: 543-546 (1995); Pelleymounter et al. (Science 269: 540-543(1995)); and Campfield et al. (Science 269: 546-549(1995)) described the weight-reducing effects of the plasma protein OB (leptin) encoded by the obese gene. A transmembrane receptor for OB protein has been cloned (Tartaglia et al. Cell 83: 1263 (1995)). Mice with mutations in the diabetes gene db have a phenotype similar to mice having mutations in ob. Chua et al. (Science 271: 994-996(1996)) have demonstrated by genetic mapping and genomic analysis, that mouse db, rat fatty (a homologue of db), and the gene encoding OB-R are the same gene.